Bone homeostasis in the adult or aging skeleton, is perturbed by mechanisms that involve decreased defense against oxidative stress and/or increased production of Reactive Oxygen Species (ROS). Remarkably, several molecules that regulate the organism's response to oxidative stress are also shared by signaling cascades that lead to prolongation of lifespan. In bone, and in any other tissue, a variety of metabolic reactions and exogenous agents generate ROS that can damage cellular constituents. Cells counteract the adverse effects of ROS by mechanisms that involve dephosphorylation and subsequent activation of a family of ubiquitous transcription factors known as FOXOs. FOXO1, one of the three FOXO homologs, regulates cell differentiation, promotes either cell survival or apoptosis; and also increases lifespan in model biologic systems. Cellular fate in response to FOXO1 dephosphorylation depends on Sirtuins. Sirtuins are NAD-dependent protein deacetylases which attenuate stress-induced apoptosis and extend lifespan in flies, worms and mammals. The mammalian homolog SIRT1, deacetylates FOXO1, thus shifting FOXO-dependent responses away from cell death and towards cell survival. We have found that FOXO1 and SIRT1 exert direct anti-apoptotic or proliferative effects on osteoblasts and osteoblast precursors. They are also expressed in osteoclasts. SIRT1-dependent deacetylation of FOXO1 is required for its anti-apoptotic effects. Most importantly, FOXO1 haploinsufficiency decreases bone mass and compromises bone microarchitecture in adult mice. Deletion of FOXO1 from osteoblasts results in reduction in osteoblast numbers without affecting osteoclast numbers. Conversely, transgenic mice expressing SIRT1 show enhanced deacetylation of FOXO1 in bone, increased bone mass in the spine and femur and increased osteoblast but decreased osteoclast numbers. Finally, FOXO1 physically associates with -catenin, a key component of the Wnt signaling pathway, to form a functional complex. Oxidative stress increases FOXO1-mediated transcription of anti-oxidant enzymes; and attenuates both the anti-osteoclastogenic and the osteoblastogenic effects of -catenin-mediated transcription in cells of the osteoblastic lineage. In studies to be conducted in this proposal, we will test the hypothesis that SIRT1/FOXO1 signaling is activated in response to physiological levels of oxidative stress to protect bone mass and preserve bone homeostasis. An interaction between this pathway and -catenin may enhance FOXO1-mediated transcription and/or regulate the anti-osteoclastogenic properties of -catenin. In this proposal we will determine the role of FOXO1 in osteoblast function. We will also elucidate the role of SIRT1 by itself or as an activator of FOXO1 signaling in bone. Finally, we will examine whether FOXO1 regulates the anti-osteoclastogenic actions of -catenin. These studies will provide for the first time a link between pathways that regulate oxidative stress, longevity and skeletal homeostasis under the control of the Sirtuin/FOXO system. Treatment modalities aimed at restoring FOXO deacetylation and phosphorylation may form the basis for a novel approach to osteoporosis therapy. PUBLIC HEALTH RELEVANCE. In bone, and in any other tissue, a variety of metabolic reactions and exogenous agents generate Reactive Oxygen Species (ROS) that can damage cellular constituents. Cells counteract the adverse effects of ROS by mechanisms that involve the NAD-dependent protein deacetylase SIRT1 and the transcription factor FOXO1; both of which extend lifespan in flies, worms or mammals. We will test the hypothesis that SIRT1/FOXO1 signaling is activated in response to physiological levels of oxidative stress to protect bone mass and preserve bone homeostasis. An interaction between this pathway and 2-catenin may enhance FOXO1-mediated transcription and regulate the anti-osteoclastogenic properties of 2-catenin.